(I) The Rel Family
c-Rel, cloned by Dr. Howard Temin's group in the 1980's, is the cellular homolog of the v-Rel oncogene encoded by the avian REV-T retrovirus. Subsequent cloning of NF-kB, p50 (NF-kB1) and p65 (RelA), in the early 1990's by Dr. David Baltimore's group identified the homology between NF-kB and c-Rel at the Rel Homologous Domain (RHD). Two other genes containing the RHD, p52 (NF-kB2) and RelB, were also identified by several groups. Hence, these five proteins are classified as the Rel transcription factor family. NF-kB and c-Rel are regulated by the “classical” pathway via the IKKα/β/γ kinase complex, whereas RelB and p52. (NF-kB2) are regulated by the “alternative” pathway via the IKKα/NIK. Despite the similarity, each Rel member is distinct with regard to tissue expression pattern, response to receptor signals, and target gene specificity. These differences are evident from the non-redundant phenotypes exhibited by individual Rel knockout mouse. Thus, therapeutics targeted to different Rel members have different biological effects and safety/toxicity profiles.
C-Rel is distinct from NF-kB (p50, p65). c-Rel is the cellular homolog of the v-Rel oncogene encoded by the avian REV-T retrovirus. Unlike the NF-kB p50 and p65 that are ubiquitously expressed in all of the cells of the body, c-Rel is exclusively expressed in cells of hematopoietic origin including T cells, B cells, macrophages, and dendritic cells. In addition, c-Rel and NF-kB regulate distinct sets of target genes in different cells. As a result, they have distinct biological functions. c-Rel is a key culprit in many of the inflammatory and autoimmune diseases.
Many receptors and stimuli can activate Rel, including TCR/BCR, TNF receptor superfamily (e.g. CD40, TNFR1, TNFR2, BAFF, APRIL, RANK), the IL-1/TLR receptors, and the Nod-like receptors, as well as activating oncogenes (e.g. Src, Ras, LMP-1, Tax, v-FLIP), reactive oxygen radicals, radiation, and chemotherapeutic agents. In response to these stimuli, Rel regulates the expression of cytokines, chemokines, adhesion molecules, costimulatory molecules, cell cycle molecules, anti-apoptotic proteins, and angiogenic factors. As such, Rel transcription factors are important therapeutic targets for many human disorders, including inflammation, autoimmune diseases, and cancer.
Many human diseases including inflammation, autoimmune disease, and cancer are attributed to aberrant activation of transcription factors, which leads to dysregulated target gene expression and evidence of new biological activities as well as survival or proliferative advantages. In the transcription factor field, NF-kB has attracted central attention as being a transcription factor that is involved in a myriad of biological functions and pathological conditions including the regulation of innate and adaptive immune response to infection, inflammation, cell survival, and tumorigenesis.
Anti-inflammatory and immunosuppressive therapies for inflammation, autoimmune disease, and transplantation have undergone revolutionary development in the past several decades. Early therapies for treating the symptoms of autoimmune/inflammatory disorders relied on glucocorticoids or corticosteroids, hormones from the adrenal medulla discovered in the 1950's. Glucocorticoids are known to be effective in dampening the signs and symptoms of inflammation and the resultant immunopathology in many inflammatory disorders, including rheumatoid arthritis, asthma, allergic dermatitis, inflammatory bowel disease, multiple sclerosis, transplant rejection, graft vs host (GvH) disease, and organ-specific autoimmune diseases, such as thyroiditis and diabetes. Unfortunately, corticosteroids cause severe systemic side effects that impact almost all organ systems, and which preclude their chronic administration.
Palliation of the symptoms of chronic inflammatory disorders such as rheumatoid arthritis is made possible by drugs classified as non-steroid anti-inflammatory drugs (NSAIDs). However, long-term use of many of these agents can cause gastrointestinal (GI) bleeding. In the 1990s, a new class of drugs known as selective inhibitors of Cox2 (Vioxx®, Celebrex®, Bextra®) was developed to treat pain and inflammation but circumventing the NSAID's side effects on the GI tract. Both NSAID and Cox2 inhibitors generally treat only symptoms and relieve pain for autoimmune patients; these drugs are generally unable to curb the progression of the disease. Moreover, the sale of Cox2 inhibitor drugs declined significantly as cardiovascular risks appeared to be common in this class of drugs.
In the 1990's, novel biologics that block tumor necrosis factor (TNF), an inflammatory cytokine, were developed. The three drugs in this class, Enbrel®, Remicade®, and Humira®, have had a major impact in slowing the joint damage caused by rheumatoid arthritis, and one of the drugs is also approved to treat psoriasis, Crohn's disease, and ankylosing spondylitis. While these new biologics drugs have fewer side effects than steroids, they are generally very expensive and may be associated with risk of infections and certain cancers. Moreover, 30-35% of patients tend to become refractory to anti-TNF therapy over time due to the production of neutralizing antibodies.
These facts make apparent the need for alternative safe and efficacious therapies that are also affordable for the treatment of inflammatory, autoimmune, and related diseases and conditions. As suggested by the success of the TNF-blocking class of drugs, a therapy that targets specific cellular proteins involved in the core disease mechanism of autoimmunity is most desirable since such a therapy will slow disease progression. Based on the fundamental function of c-Rel in immune cells, c-Rel blockade further finds use in the treatment of other pathological conditions including inflammation, autoimmune disease, bone loss, transplant rejection, lymphoma, and solid tumors.
Cancer remains an incurable disease. Most current cancer therapies such as chemotherapies have broad cellular targets and exhibit unbearable side effects on the patients. The success of Gleevec® in CML and other related cancers has proved the principle that targeted therapy can be achieved as long as the oncogenic target is identified. c-Rel was first characterized as a proto-oncogene in chicken. Subsequently, c-Rel gene amplification or constitutive activation has been documented in many human B cell leukemia, lymphoma, as well as tumors derived from solid tissues. Therefore, c-Rel is a novel therapeutic target for human cancers with over-reactive c-Rel or NF-kB activity.
(II) c-Rel Knockout Mouse Studies Validate c-Rel as a Drug Target for Inflammatory and Autoimmune Diseases
Evidence from knockout animal models and human genetic association studies support c-Rel as a potential therapeutic target for inflammatory and autoimmune diseases. Using c-Rel knockout mice, the Liou laboratory first showed that blocking c-Rel protected mice from developing experimental autoimmune encephalomyelitis (EAE) and Streptozocin-induced diabetes (Hilliard, B A et.al. 2002. J. Clin. Inv. 110, 843; Lamhamedi-Cherradi, S et. al. 2003. J. Immunol. 171,4886). Subsequent studies by us and others further demonstrated the role of c-Rel in collagen-induced arthritis, allergic asthma, Helicobacter hepaticus-induced colitis, CCl4-induced liver inflammation, and stress-induced atherosclerosis (Campbell, I et. al. 2000. J. Clin. Inv. 105, 1799; Finn P W et. al. 2001. J. Immunol. 167, 5994; Finn P W et. al. 2002, J. Leuk. Biol., 72, 1054; Yang H. et. al. Transplantation, 2002. 74, 291; Wang, I et. al. 2008. J. Immunol. 180, 8118).
At cellular and molecular levels, c-Rel contributes to multiple steps in autoimmune diseases. These include inducing the expression of inflammatory cytokines of the Th1 and Th17 immune responses (e.g. IL-2, IFN-γ, TNF, IL-12/IL23 members), costimulatory function of antigen presenting cells (e.g. IL-12/IL23 members, OCILRP2), activation of autoreactive lymphocytes (via cell cycle and cell survival proteins), and antibody production. These collective studies thus validated c-Rel as a potential novel therapeutic target for autoimmune diseases.
In addition, intriguing data from recent large-scale genome-wide association studies link several genes in the Rel pathways with increased risks in human autoimmune diseases. These include the association of CD40, c-Rel, Btk, Blk, PKCθ, A20, and TRAF1 genetic variants with rheumatoid arthritis (Criswell, L A et. al. 2010. Immunol. Rev. 233, 55). Previous genetic linkage studies have also identified IL-2/IL2Rα (CD25) and CTLA4 variants as risk markers for Type 1 diabetes, Grave's disease, and inflammatory bowel disease and also link CTLA4 and PTPN22 genetic variants with many autoimmune diseases (Marquez, A et. al. 2009. Am. J Gastroenterol. 104, 1968; Glas J et. al. 2009. Am. J Gastroenterol. 104, 1737). It is important to note that these risk factor genes functionally converge at the Rel transcription factors, including the receptors (CD40, CTLA4), signaling molecules (e.g. Btk, PKCθ, TRAF1), and its downstream targets (e.g. IL-2, CD25, A20), thus corroborating the fundamental role c-Rel in the pathogenesis of autoimmune diseases in general.
Autoimmune diseases arise from the host immune system attacking its own tissues. There are at least 80 autoimmune diseases afflicting various tissues such as joints (rheumatoid arthritis), the central nervous system (multiple sclerosis), intestines (Crohn's disease), or the skin (psoriasis). It is estimated that autoimmune diseases affect 5-8% of the American population, or approximately 23.5 million people. Since the underlying mechanisms of autoimmune diseases are similar, the Rel inhibitors described in this invention are applicable for the treatment of most of human autoimmune diseases, as listed in Table 2.
Anti-inflammatory and immunosuppressive therapies for inflammation, autoimmune disease, and transplantation have undergone revolutionary development in the past several decades. Since the 1950's, glucocorticoids have been widely used in dampening the signs and symptoms of inflammation and the resultant immunopathology in almost all inflammatory disorders, including rheumatoid arthritis, asthma, allergic dermatitis, inflammatory bowel disease, multiple sclerosis, transplant rejection, graft vs. host (GvH) disease, and organ-specific autoimmune diseases such as thyroiditis and diabetes. It has been shown that the primarily anti-inflammatory activity of glucocorticoids is through the inhibition of Rel activity. Unfortunately, corticosteroids have other cellular targets. Long-term use of corticosteroids can cause severe systemic side effects that impact almost all organ systems, and which preclude their chronic administration. Thus, the euphoria that corticosteroids might be “the cure” for chronic autoimmune and inflammatory diseases rapidly dissipated even before the 1960s. Subsequent development of non-steroid anti-inflammatory drugs (NSAIDs) and Cox2 inhibitors only treat symptoms and relieve pain for autoimmune patients. These drugs, however, are unable to curb the progression of the disease process. Long-term use of NSAIDs can cause gastrointestinal (GI) bleeding, whereas the Cox2 inhibitors were found to associate with increased cardiovascular risks.
Currently, there are several biologics based therapies for autoimmune diseases. The most successful agents are a new class of biologics that block TNF, e.g., Enbrel®, Remicade®, and Humira®. While these new biologic drugs are effective for the treatment of rheumatoid arthritis, psoriasis, and Crohn's diseases, 30-35% patients become refractory to anti-TNF therapies over time due to the production of neutralizing antibodies. Thus, there remains an unmet medical need for anti-TNF resistant patients.
Anti-TNF therapies, however, have not yet shown therapeutic effects on multiple sclerosis (MS). MS patients with relapsing remitting diseases are currently treated with a few disease-modifying drugs, including β-IFNs (Betaseron®, Avonex®, Rebif®), glatiramer acetate (Copaxone®), and Natalizumab (Tysabri®). These drugs are generally ineffective for primary progressive or secondary progressive MS patients. Unfortunately, most patients treated with these drugs eventually relapse and develop disease progression. In addition, Tysabri® has safety concerns as it may increase the risk of progressive multifocal leukoencephalopathy (PML) in small percentage of patients with MS, Crohn's disease, and psoriasis. In 2010, the FDA approved a new oral drug Fingolimod (Gilenya®) for the treatment of relapsing remitting MS patients. Fingolimod targets lysophospholipid S1P1 receptors and prevents lymphocyte migration into CNS. Post-marketing collection of data will help evaluate its safety profile and therapeutic superiority in larger patient pools.
Other therapies currently under clinical trials for autoimmune diseases, which also intercept the Rel pathway, include anti-CD20 (approved for rheumatoid arthritis; clinical trial for multiple sclerosis), anti-IL12/IL23 (approved for psoriasis; clinical trials for Crohn's disease, psoriatic arthritis), anti-IL17 (clinical trials for RA, Crohn's disease, psoriasis, psoriatic arthritis, uveitis), anti-IL-6 (approved for RA, juvenile RA, Crohn's disease, Castleman's disease; clinical trials for other autoimmune disorders, multiple myeloma, prostate cancer), and anti-IL1 (approved for RA, cryopyrin-associated periodic syndrome; clinical trials for RA, juvenile RA, COPD, gout, type 2 diabetes, coronary atherosclerosis).
In conclusion, many autoimmune diseases including multiple sclerosis, ankylosing spondylitis, and type 1 diabetes still have no effective treatments. Existing biologic drugs are very expensive and require administration by injection, thus reducing patient compliance. Therefore, there is a need for identifying new Rel inhibitors and validating their therapeutic potential in autoimmune diseases.
(III) Rel and Tumorigenesis
Many studies, including those from the inventor's lab, have reported the association of hyperactive Rel with human cancers. This may come as no surprise, as several molecules in the Rel pathways were initially identified as potential oncogenes. For example, c-Rel gene amplification and the p52 (p100, lyt10) gene truncation were frequently found in DLBCL. The Rel family has been shown to regulate the expression of cell cycle regulators, anti-apoptotic proteins, inflammatory mediators, cytokines, growth factors, chemokines, and adhesion molecules. As such, Rel could participate in various aspects of tumorigenesis including tumor growth, survival advantage, chemoresistance, angiogenesis, and metastasis. A review of the involvement of Rel in a variety of tumors and the potential mechanism involved in the tumorigenesis follows.
For many virus-induced tumors, it is well-established that some viral oncogenes can directly activate the Rel signaling pathways. For example, in HHV8 (or KSHV)-induced primary effusion lymphoma, it has been shown that the viral oncogene vFLIP associates with TRAFs signaling molecules, leading to constitutive activation of NF-kB (Guasparri I et. al. 2006. EMBO 7, 114). In Burkitt's lymphoma, EBV viral protein LMP-1 also works in a similar mechanism by associating with TRAFs, thus activating signaling pathways normally activated by the TNF receptor members such as CD40 and receptors for Baff and April. The Tax oncoprotein, expressed by HTLV-1 that induces adult T cell leukemia, is shown to activate the Rel pathway by binding to the IKK complex.
Rel activation has been reported in most B cell tumors, including multiple myeloma, diffuse large B cell lymphoma, CLL, primary mediastinal lymphoma, Burkitts' lymphoma, mantle cell lymphoma, MALT lymphoma, and Hodgkin's diseases (See Table 2). For many B cell tumors, the persistent activation of Rel family has been attributed to mutations in the Rel signaling pathways or overexpression of Rel activators. For example, it has been shown that some multiple myeloma (MM) cells have overexpression of the positive regulators of the NF-kB pathway (e.g. CD40, TACI, NIK, NFKB1, NFKB2), whereas others have deletions or mutations in the negative regulators of the Rel signaling components (e.g. TRAF3, CYLD, cIAP1/2) (Annunziata C M, et. al. 2007. Cancer Cell 12,115; Keats, J. et. al. 2007. Cancer Cell 12, 131).
Similar findings were also reported in DLBCL in that mutations in multiple Rel upstream regulators were detected (e.g. A20, CARD11, TRAF2, TRAF3, TAK1, RANK) (Compagno M et. al. 2009. Nature 459(7247):717; Bidère N et. al. 2009. Nature 458, 92).
In CLL however, the survival of tumor cells and its constitutive Rel activity is mostly attributed to persistent activation of the CD40 and the B cell antigen receptor (BCR) signaling pathways, rather than mutations in the signaling pathways (Furman, R R et. al. 2000. J. Immunol. 164, 2200; Bernal, A, et. al. 2001. Blood 98, 3050).
The Rel (NF-kB) has also been shown to be involved in epithelial derived solid tumors. Earlier studies in the late 90's have shown that NF-kB is required for Ras and Bcr-Abl mediated tumorigenesis. Subsequently, several studies point to the involvement of Rel activation in breast tumorigenesis. First, it was shown that EGF receptors such as Her2 can activate NF-kB. A transgenic mouse model demonstrated that overexpression of v-Rel in breast epithelial cells led to the development of breast tumors. IKKε was found to be amplified or overexpressed in breast cancer cell lines and patient-derived tumors. IKKε can activate c-Rel.
Perhaps the most important theme surrounding Rel mediated tumorigenesis is the production of inflammatory mediators. Initial activation of Rel by oncogenes in tumor cells leads to the production of inflammatory mediators (e.g. IL-6, chemokines) that increase tumor survival as well as recruiting bone marrow derived immune cells. The immune cells further produce cytokines and growth factors that amplify and promote tumor cell growth, angiogenesis, and metastasis, as well as conferring drug resistance. This theme has been demonstrated in numerous tumor models (Ammirante, M et. al. 2010. Nature 464, 302; Bromberg, J et. al. 2009 Cancer Cell 15, 79; Boehm, J S et. al. 2007. Cell 129, 1065; Grivennikov, S I and Karin, M 2010. Curr. Opin. Genet. Dev. 20, 65).
For example, in a prostate cancer mouse model, it was shown that B cells and bone marrow derived cells can produce IL-6 and LTβ, which are essential for promoting prostate cancer growth after androgen deprivation. In breast cancer, increased IL-6 expression is associated with metastasis and poor prognosis. It has been shown that Rel and Stat3 synergistically regulate IL-6 expression, thus establishing a positive feedback loop in breast tumorigenesis. In colitis-associated cancer and hepatocellular carcinoma models, both IL-6 and TNF produced by bone marrow derived myeloid cells were shown to promote tumor cell growth and survival. In head and neck squamous cell carcinomas, Rel activates the expression of pro-inflammatory and pro-angiogenic cytokines IL-1α, IL-6, IL-8, and GM-CSF, which promote tumor growth in vivo.
The theme also extends to B cell tumors and other cytokines besides IL-6 and TNF. For examples, in multiple myeloma, IL-17, Baff, and April have been shown to provide autocrine and paracrine growth and survival mediated by the interaction between tumor and stromal cells. IL-23 p19 was shown to be significantly upregulated in majority of carcinoma samples from various organ types, including colon, ovarian, head/neck, lung, breast, stomach, and melanoma. The above studies thus point to potential therapeutic benefits of blocking Rel and its downstream inflammatory mediators for the treatment of a wide variety of solid tumors and blood cancers.
Emerging studies have also demonstrated that radiation therapy and many clinically used chemotherapeutic agents (e.g. doxorubicin, vinca alkaloids, vincristine and vinblastine, camptothecin), can actually induce Rel activity. While some cancer therapies, such as Velcade® and thalidomide, presumably work through inhibiting Rel activity, recent studies have shown that resistance to these drugs is associated with increased Rel activation. Thus, it is conceivable that Rel inhibitors might provide therapeutic benefits to cancer patients either as monotherapy or combination therapy with other cancer drugs.
(IV) Other Diseases Associated with Rel Activation
Rel activation has also been implicated in a wide variety of diseases and pathological conditions, including AIDS, diabetes mellitus, cardiovascular diseases, atherosclerosis, septic shock syndrome, viral replication, osteoporosis, bone loss, organ transplant rejection, graft-versus-host diseases (GVHD), neurodegenerative disorders, ataxia telangiectasia, metabolic disorders, type 1 and type 2 diabetes, as well as aging. Specifically, the c-Rel knockout mice studies have clearly demonstrated the involvement of c-Rel activation in stress-induced atherosclerosis (A. Bierhaus et. al. 2010. JCI) and transplant rejection (Finn P W et. al. 2001, J I; Finn, P W et. al. 2002, J Leukoc. Biol; Yang H. et. al. Transplantation, 2002).